The Role of Aromatic Residues In the Catalytic Domain Tunnel of Hypocrea Jecorina Cellobiohydrolase Cel6A

The glycoside hydrolase Family 6 cellobiohydrolase (Cel6A) from Hypocrea jecorina – formerly Trichoderma reesei – plays an important role in the enzymatic cocktail used to breakdown cellulosic feedstock to soluble sugars.  The catalytic domain of this enzyme includes an enclosed tunnel consisting of four structurally-defined binding sites through which cellulose threads and is hydrolyzed to cellobiose or glucose.  Along the length of the tunnel, aromatic residues assist in glucose ring binding via stacking interactions.  We use molecular simulation and free energy calculations – specifically, thermodynamic integration – to provide insight into how mutations to the aromatic residues in the active site tunnel affect enzymatic processivity, protein structure, and the conformation of the catalytically-active complex.  As aromatic residues are ubiquitously found in the tunnels of carbohydrate-active enzymes, these results have implications for understanding protein-ligand binding for general carbohydrate turnover in the biosphere and in protein engineering for enhanced conversion rates.